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(American Journal of Pathology. 2005;166:1773-1779.)
© 2005 American Society for Investigative Pathology

Absence of Immunoglobulin Class Switch in Primary Lymphomas of the Central Nervous System

Manuel Montesinos-Rongen^*, Roland Schmitz, Cornelius Courts^*, Werner Stenzel^*, Dörte Bechtel, Gerald Niedobitek, Ingmar Blümcke, Guido Reifenberger^¶, Andreas von Deimling^||, Berit Jungnickel^**, Otmar D. Wiestler, Ralf Küppers and Martina Deckert^*

From the Department of Neuropathology,^* University of Cologne, Köln; the Institute for Cell Biology (Tumor Research), University of Essen, Medical School, Essen; the Institute for Pathology and Department of Neuropathology, Friedrich-Alexander-University, Erlangen; the Department of Neuropathology,^¶ Heinrich-Heine-University, Düsseldorf; the Department of Neuropathology,|| Charité, University Medicine, Berlin; the Institute of Clinical Molecular Biology and Tumor Genetics,^** GSF, München; and the German Cancer Research Center, Heidelberg, Germany

	Abstract

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Primary lymphomas of the central nervous system (PCNSLs) were investigated for their capacity to perform further maturation steps. We studied a series of 11 PCNSLs derived from immunocompetent patients for immunoglobulin (Ig) class switch recombination (CSR) by performing reverse transcriptase-polymerase chain reaction (RT-PCR) for transcripts of Ig constant region gene segments (IGHC). This analysis revealed exclusive transcription of IgM and IgD mRNA in the absence of IgG, IgA, or IgE transcription. This finding was corroborated at the protein level by the immunohistochemical demonstration of IgM on the surface of the tumor cells. The unexpected lack of CSR may be due to internal switch µ region deletions, which were detected in 7 of 11 cases. We also found that expression of activation-induced cytidine deaminase (AID), which is required for CSR and somatic hypermutation, was detectable by RT-PCR in 4 of 10 cases and by immunohistochemistry in one of three cases analyzed. This may indicate that ongoing somatic mutation, which is often observed in PCNSL, could be due to sustained AID expression in a fraction of cases and that intraclonal V gene diversity may occur in other cases at an earlier phase of tumor clone expansion, when AID may have been expressed.

These observations raise the question of whether the tumor cells of PCNSL may perform further maturation steps, including Ig class switch recombination (CSR). CSR replaces the µ constant region with one of the downstream-located constant regions, allowing the generation of different antibody classes.⁹ CSR occurs within the 3- to 5-kb repetitive switch region sequences located 5' of each constant region gene segment (IGHC). Although the precise molecular mechanisms of CSR have not yet been fully elucidated, the essential and sole (human) B-cell-specific known factor required for CSR is the enzyme activation-induced cytidine deaminase (AID).^10,11

In AID-deficient mice, both CSR and somatic hypermutation (SHM) are abolished, illustrating that this protein plays a pivotal role in both processes, thus, closely linking SHM and CSR.¹² The pattern, distribution, and levels of AID protein in various B-cell subsets and B-cell neoplasms have only incompletely been determined. RNA transcription analysis indicated a selective expression of AID in GC B cells and in follicular lymphoma and DLBCL, which are GC B-cell-derived lymphomas.^11,13,14 However, the expression of AID in PCNSL has not yet been analyzed. The aim of the present study was to clarify whether tumor cells of PCNSL had undergone CSR and to assess AID mRNA and protein levels in this CNS-specific subtype of DLBCL.

	Materials and Methods

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Histopathology

Stereotactic tumor biopsies of 11 immunocompetent patients (five female and six male; mean age, 62; range, 28 to 75 years) with a histopathologically confirmed diagnosis of PCNSL were analyzed. All studies were approved by local Ethics Committees; informed consent was provided according to the Declaration of Helsinki. Systemic lymphoma manifestation was excluded by extensive staging. Human nonmalignant tonsils were obtained with written consent from patients undergoing tonsillectomy. All tumors were histopathologically classified as DLBCL according to the World Health Organization classification.² The diagnoses were based on a combination of routine morphology and immunohistochemistry with antibodies against Ki-67 (clone MIB-1; Dako, Hamburg, Germany), CD45 (clone T29/33; Dako), CD3 (polyclonal; Dako), and CD20 (clone L26; Dako), as reported before.⁴ In addition, immunohistochemical analyses were performed with monoclonal mouse antibodies against IgM (clone R1/69; Dako) and IgG (clone A57H; Dako). An ABC protocol was applied using 3,3'-diaminobenzidine (Sigma, Deisenhofen, Germany) as chromogene and H₂O₂ as co-substrate. Expression of AID was investigated in three PCNSLs (cases 6, 10, and 11) using the monoclonal rat anti-human AID antibody EK2-5G9 (IgG_2b).¹⁵ Briefly, frozen sections were fixed in 4% paraformaldehyde and subjected to antigen retrieval as described previously¹⁵ before application of the primary antibody. Bound antibody was detected using an APAAP protocol with rabbit anti-rat IgG and rat APAAP (both from Dakocytomation, Hamburg, Germany).

Isolation of GC B Cells

Human tonsils were minced and mononuclear cells were obtained through a Ficoll density gradient (Amersham Biosciences, Freiburg, Germany). B cells were enriched with CD19-coupled magnetic beads (Miltenyi, Bergisch-Gladbach, Germany). Thereafter, GC B cells were stained with monoclonal mouse anti-human CD38-PE (BD, Heidelberg, Germany) and CD77-FITC (BD). Centrocytes and centroblasts were sorted as CD38⁺CD77^– and CD38⁺CD77⁺ cells, respectively, using a FACS Vantage cell sorter (BD).

DNA Extraction

DNA was extracted from frozen tissue blocks harboring at least 80% tumor cells with the NucleoSpin Tissue kit (BD Clontech, Heidelberg, Germany). DNA was dissolved in 100 µl of TE buffer (pH 7.6). Two to 5 µl of this stock DNA solution, corresponding to 100 ng of DNA, was used in each amplification reaction.

RNA Extraction

RNA was extracted from frozen tumor tissue blocks, which contained at least 80% tumor cells, either with the SNAP total RNA isolation kit (Invitrogen, Karlsruhe, Germany) or using the trizol/chloroform method. First-strand cDNA was either synthesized using the SuperScript II kit (Invitrogen) or using the High Capacity cDNA Archive kit (Applied Biosystems, Weiterstadt, Germany).

Polymerase Chain Reaction (PCR) Analysis

Rearrangements of the immunoglobulin heavy (IgH) chain locus were amplified with VH gene segment family-specific primers from genomic DNA as published previously.⁴ Ten microliters of each PCR reaction was analyzed on a 2% agarose gel stained with ethidium-bromide. PCR products were extracted from a 2% low-melting agarose gel (NuSieve GTG Agarose; Biozym, Hessisch-Oldendorf, Germany) using the QIAEx II kit (Qiagen, Hilden, Germany) and directly sequenced from both sides using the BigDye kit (Applied Biosystems) and an ABI377 automated sequencer (Applied Biosystems). Sequences were compared with human germline Ig gene sequences using the IMGT¹⁶ and GenBank databases and DNAPlot, MacVector, and Blast software.

The switch µ (Sµ) region was amplified using IgMrev, 5'-TCG TAT CCG ACG GGG AAT TCT CAC AG-3', and Sµfor, 5'-TCC ATC CAG CTT TCA GAA ATG GAC TC-3', as reverse and forward primers, respectively, applying the GeneAmp XL PCR system (Applied Biosystems) supplemented with 0.75 mmol/L Mg(OAc)₂ and 1.5x PCR Enhancer solution (Invitrogen). Cycling conditions were 95°C for 5 minutes, 44 cycles at 95°C for 60 seconds, 62°C for 45 seconds, and 72°C for 6 minutes, followed by a final extension step at 72°C for 15 minutes. Two PCR products were isolated from agarose gels and directly sequenced from both sites using the BigDye kit.

Reverse Transcriptase (RT)-PCR Analysis

All cDNAs were tested by RT-PCR for transcripts of ß-actin as housekeeping gene (accession no. nm_001101). As primers, BA-F, 5'-TTT CTT GAC AAA ACC TAA CTT GCG-3', and BA-R, 5'-TAG GAT GGC AAG GGA CTT CTT G-3', were used, amplifying a 266-bp product (position 1231–1496). Amplification was carried out for one cycle at 95°C for 5 minutes, 60°C for 30 seconds, and 72°C for 1 minute, followed by 24 cycles at 95°C for 50 seconds, 60°C for 30 seconds, and 72°C for 1 minute. A final extension step at 72°C for 10 minutes was added. Standard TaqDNA polymerase (Invitrogen) was added before PCR. PCR reactions without DNA served as negative control.

Lymphoma-derived cDNAs were tested by PCR for transcripts of IGHC gene segments. A clone-specific forward primer, corresponding to a fragment of the complementarity-determining region 3 (CDR3; Table 1 ), was used together with an isotype-specific reverse primer in five different PCRs. For isotype IgM: IGHC-M, 5'-GTG GGA CGA AGA CGC TCA CTT TGG-3'; for isotype IgD: IGHC-D, 5'-CTG GCC AGC GGA AGA TCT CCT TC-3'; for isotype IgG1, IgG2, IgG3, and IgG4: IGHC-G, 5'-CTT GTC CAC CTT GGT GTT GCT GG-3'; for isotype IgE1 and IgE2: IGHC-E, 5'-GAC GAC TGT AAG ATC TTC ACG GTG-3'; and for isotype IgA1 and IgA2: IGHC-A, 5'-ACA GTC ACA TCC TGG CTG GRA TTC-3' were applied, respectively. Amplification was carried out for one cycle at 95°C for 3 minutes, 57°C for 30 seconds, and 72°C for 1 minute, followed by 34 cycles at 95°C for 30 seconds, 57°C for 30 seconds, and 72°C for 1 minute. A final extension step at 72°C for 10 minutes was added. Platinum TaqDNA polymerase (Invitrogen) was added before PCR. As positive controls, cDNAs derived from human tonsils and various Epstein-Barr virus-transformed B-cell lines (J8 and J17) were used; as negative controls, HeLa cell line-derived cDNA and water instead of cDNA were used, respectively.

	Results

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The data for IgH rearrangements detected in the 11 PCNSLs analyzed are summarized in Table 1 . All tumors carried potentially functional immunoglobulin heavy chain variable gene segment (IGHV) gene rearrangements. Forty-five percent (5 of 11) of the tumors used an IGHV of the VH3 gene family, whereas the remaining 55% (6 of 11) rearranged a gene segment of the VH4 gene family (Table 1) , with five of these six PCNSLs having rearranged the IGHV4–34 gene segment. This preferential usage of the V4–34 gene segment in PCNSL confirms earlier data.^4,5 The mean mutation frequency of this series was 13.9%, with a range from 4.1 to 23.2%. All rearrangements were in-frame, and stop-codons were not identified. Thus, all rearrangements were considered as potentially functional. Sequences of the IgH rearrangements derived from patients 1 to 11 are available through GenBank accession nos. AF168818 and AY598953 to AY598962.

To determine the Ig isotype of the tumor cells, individual clone-specific primers, localizing to the CDR3, were designed for each tumor and used together with primers specific for the various constant region genes. RT-PCR detected IgM and IgD mRNA in all tumors, whereas no IgG, IgA, or IgE mRNA transcripts were expressed in any of the 11 PCNSLs (Table 1) . This observation was confirmed and extended at the protein level: immunohistochemistry revealed IgM expression on the surface of the tumor cells in all PCNSLs (Figure 1, a and b) , whereas IgG protein was consistently absent from the malignant cells (Figure 1, c and d) .

View larger version (153K): [in this window] [in a new window]   Figure 1. Immunohistochemical phenotype of PCNSL. a and b: Strong IgM expression on the surface of the tumor cells in a PCNSL (a) as well as in the GC of a tonsil (b). Anti-IgM immunostaining, slight counterstaining with hemalum, x200. c and d: Tumor cells do not express IgG (c), whereas IgG staining is visible in a tonsillar GC (d). Anti-IgG immunostaining, slight counterstaining with hemalum, x200. e and f: AID can be detected in some tumor cells of a cerebellar tumor (e) but is not expressed in PCNSL in characteristic supratentorial location (f), x100. Inset I: negative control with omission of the primary antibody; inset II: positive control with AID expression in the GC of a tonsil, x100.

View larger version (18K): [in this window] [in a new window]   Figure 2. AID mRNA transcription in PCNSL. Quantitative RT-PCR analysis of AID normalized to RNA polymerase II expression and relative to centroblasts (CB). In PCNSL (open bars), AID mRNA levels are generally low compared with centroblasts (CB) and centrocytes (CC). Only one PCNSL (no. 4), which was located in the cerebellum, transcribed increased levels of AID mRNA. B, Raji cell line-derived cDNA; H, HeLa cell line-derived cDNA. The CT method was used. Normalization to TATA box binding protein yielded identical results (data not shown). Samples were analyzed in triplicate.

View larger version (82K): [in this window] [in a new window]   Figure 3. Internal deletions in the switch µ region of PCNSL. A: PCR amplification of the Sµ region of nine PCNSLs (lanes 2 to 10), Jurkat DNA as a positive control for the full-length product (lane 11), water control (lane 12), and 1-kb DNA ladders (lanes 1 and 13). All PCNSLs exhibited amplification of the full-length product (4.5 kb), whereas in four cases, one additional band (marked with asterisks) and in one case two additional bands were observed. B: Graphical representation of switch µ region flanked by the JH6 gene segment, the intronic IgH enhancer, and the Cµ gene. Arrows indicate the primers used for amplification. The shorter products obtained from PCNSL cases 2 and 5 were sequenced. These show deletions of 3.3 kb (case 5; lane 5 in A) with a single breakpoint and 1.8 kb (case 2; not shown in A) with multiple breakpoints in the switch µ region. Due to the repetitive structure of the switch µ region the short sequence fragments of case 2 cannot be unequivocally assigned to definitive positions. Sequences are available from GenBank under accession numbers AY337410 and AY337411.

	Discussion

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Lack of CSR was documented by both RT-PCR and immunohistochemistry, demonstrating an IgM/IgD phenotype and an expression of IgM on the surface of the tumor cells in all 11 PCNSLs of this series. Remarkably, AID mRNA, a prerequisite for CSR, was expressed at levels comparable with or even exceeding levels detected in normal GC B cells in 4 of 10 cases analyzed. Immunohistochemistry in three cases paralleled the quantitative RT-PCR results, showing absence of AID protein in two RT-PCR-negative cases and presence of AID protein in one of the RT-PCR-positive cases.

In light of the high somatic mutation load but retained IgM and IgD expression in PCNSL, there appears to be a dissociation between CSR and SHM activity in this lymphoma entity. Interestingly, in extracerebral DLBCL, expression of AID was not associated with an ongoing IgV hypermutation activity, which is also a characteristic feature of PCNSL,⁸ because subcloning experiments failed to show intraclonal heterogeneity even in DLBCL displaying the highest AID protein amounts.¹⁸ Furthermore, there was no correlation between AID protein levels and the mutation load in extracerebral DLBCL with aberrant SHM of the PIM1, PAX5, RhoH/TTF, and C-MYC genes.¹⁸ Collectively, these data indicate a poor correlation between SHM and AID expression and raise the questions 1) whether low levels of AID may be sufficient for induction and maintenance of SHM but not for CSR; 2) whether SHM and intraclonal diversity have resulted from an earlier phase of clonal expansion, during which the tumor cells have expressed AID, which has been downregulated at later stages of tumor development; and 3) whether other genetic events may have rendered the tumor cells of PCNSL independent of AID.

What might be the reason for the lack of CSR in PCNSL? First, atypical rearrangements involving downstream CH genes have been described in B-cell lymphomas and may prevent normal CSR. In IgM-expressing follicular lymphoma, which are also characterized by highly mutated Ig genes as well as ongoing mutation, aberrant CSR events between downstream CH genes with retention of Cµ and C were frequent.¹⁹ However, other types of B-cell lymphomas lack such recombination events,¹⁹ so that it remains presently unclear whether downstream CH gene deletions may be involved in the rare occurrence or absence of PCSNL with switched isotype.

A second reason could be related to the microenvironment, which may also affect and determine isotype switch of B cells. For example, GC B cells in Peyer’s patches of the small intestine preferentially switch to IgA, whereas GC B cells in lymph nodes usually switch to IgG. In this regard, the regional cytokine milieu, in particular interleukin (IL)-4, IL-10, and transforming growth factor-ß, may play a critical role in controlling CSR.^20-23 Under physiological conditions in the CNS, only low levels of cytokines such as IL-10, but no IL-4, can be detected.²⁴ Thus, the tumor cells of PCNSL may originate from (possibly ectopic) GC, which provided support for SHM but not for isotype switch.

Third, the lack of CSR in PCNSL might reflect derivation of the malignant intracerebral B cells from a distinct subset of B cells destined not to undergo CSR in the GC. A considerable fraction of human somatically mutated (memory) B cells expresses IgM (±IgD).^6,25 Retention of IgM expression on a subset of memory B cells may be advantageous to allow a rapid reaction on further antigenic stimulation, because IgM can strongly activate the complement system. Thus, PCNSL may originate from GC B cells destined to become IgM-expressing memory B cells. Interestingly, several other B-cell lymphoma entities with somatically mutated V genes also mostly show IgM (±IgD) expression, including Burkitt lymphoma, MALT lymphoma, and extracerebral DLBCL.²⁵

Finally, we speculated that the lack of CSR in the PCNSL could be due internal deletions within the Sµ region, which have been identified in some normal and malignant IgM-expressing B cells.^17,19,26 Because the Sµ region is important for CSR, the deletions may impair CSR and hence cause isotype stabilization.¹⁷ Applying a long-distance PCR approach to amplify the Sµ region, we detected shortened products in 7 of the 11 cases analyzed. In one of the cases, two distinct shortened amplificates were obtained, suggesting internal Sµ deletions on both IgH alleles. Somewhat surprisingly, in the six other cases with internal Sµ deletions, we obtained one shortened and one full-length PCR product. We could not clarify whether in these cases the Sµ regions with internal deletions are located on the heavy chain allele expressed. It may well be that in some of the cases, the nonproductive allele is involved in chromosomal translocations targeted to the Sµ region²⁷ or that a second internal deletion could not be amplified, because it encompassed one of the primer binding sites. Interestingly, genomic deletions within the Sµ region have also been detected in IgM-expressing B-CLL and follicular lymphomas, and there is evidence that these deletions preferentially occur on the productively rearranged heavy chain allele.^19,26 This would argue in favor of the idea that also in PCNSL, the internal Sµ deletions are often located on the IgH allele expressed and, therefore, might explain the absence of CSR in such cases.

In conclusion, the present study demonstrates that PCNSLs only rarely express AID, which may indicate that the intraclonal V gene diversity observed in a fraction of cases mainly occurs in early stages of lymphoma proliferation, when AID may still have been expressed. Moreover, PCNSLs consistently express IgM (±IgD) and do not undergo CSR, although the high load of somatic V gene mutations suggests significant AID expression in the tumor precursor cells. The absence of CSR may be due to several reasons, but importantly, we detected internal Sµ deletions in 7 of 11 cases as a potential cause for impaired CSR.

	Acknowledgements

 
We thank Marek Franitza, Elena Fischer, Irmgard Henke, Elisabeth Kremmer, Stephanie Tobollik, and Christa Winkelmann for expert technical assistance.

	Footnotes

 
Address reprint requests to Martina Deckert, M.D., Department of Neuropathology, University of Cologne, Joseph-Stelzmann-Strasse 9, D-50931 Köln, Germany. E-mail: neuropatho{at}uni-koeln.de

Supported by grants from the Deutsche Krebshilfe/Dr. Mildred Scheel Stiftung für Krebsforschung (10-2153-De 1 and 70-3173-Tr3) and the Wilhelm-Sander Stiftung (2003.046.1).

Accepted for publication February 9, 2005.

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